In scanning microscopy, a sample is illuminated with a light beam in order to observe the reflected or fluorescent light emitted from the specimen. The focus of an illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors, the deflection axes usually being perpendicular to one another so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the light coming from the specimen is measured as a function of the position of the scanning beam.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam. A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels back via the beam deflection device to the beam splitter, traverses it, and is then focused onto the detection pinhole behind which the detectors are located. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers.
An arrangement for enhancing the resolution capability for fluorescence applications is known from DE 44 16 558 A1. In this, the lateral edge regions of the focus volume of the excitation light beam are illuminated with a light beam of a different wavelength (called the “stimulation light beam”) that is emitted from a second laser, in order to bring the sample regions excited there by the light of the first laser back to the ground state in stimulated fashion. Only the light spontaneously emitted from the regions not illuminated by the second laser is then detected, so that an overall improvement in resolution is achieved. The term “stimulated emission depletion” (STED) has become established for this method.
For example from U.S. 2002/0167724 A1 or from U.S. Pat. No. 6,667,830 B1 a variant of the STED-technology is known. In this variant the specimen is dyed with molecules having three electronic states including at least a ground state and in which an excited wavelength band from the first electron excited state to the second electron excited state overlaps a fluorescent wavelength band upon deexcitation through a fluorescence process from the first electron excited state to a vibrational level in the ground state. This Variant of the STED-technology is often called “up-conversion” technology.
DE 100 56 382 A1 discloses a light source for a STED microscope. The light source contains a primary light source that emits light of one wavelength, that light being split into two light beam segments using a means for spatial division.
A means for modifying the wavelength is provided in at least one of the light beam segments. This means can be embodied, for example, as an optically parametric oscillator (OPO). The light pulses of the stimulation light beam are delayed with respect to those of the excitation light beam by a time interval which is less than the lifetime of the excited state of the dye. The pulse duration of the light pulses of the stimulation light beam should be greater than the characteristic time for a decay process of the final state of the sample dye. The light pulses of the excitation light beam typically possess pulse widths in the range from sub-picosecond to several picoseconds, whereas the light pulses of the stimulation light beam have pulse widths in the range between 10 ps and several hundred picoseconds. Grating arrangements are usually used to stretch out the light pulses of the stimulation light beam in time (T. A. Klar et al., “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” PNAS, 2000, Vol. 97, 8206-8210). The time offset of the light pulses of the stimulation light beam with respect to the light pulses of the excitation light beam is brought about at present by way of a longer path length in air for the light pulses of the stimulation light beam.
The known arrangements have on the one hand the disadvantage of a very long physical length, since a light path of several meters must be provided in order to achieve sufficient delaying of the light pulses of the stimulation light beam with respect to the light pulses of the excitation light beam, and on the other hand the disadvantage that the gratings used to stretch out the pulse duration of the light pulses of the stimulation light beam largely destroy the wavefront of the stimulation light beam because of their inherently poor surface quality.
A resolution enhancement in the direction of the optical axis can be achieved, as described in European Patent EP 0 491 289 entitled “Double confocal scanning microscope,” by way of a double objective arrangement (4-pi arrangement). The excitation light coming from the illumination system is split into two sub-beams, which simultaneously illuminate the sample as they extend toward one another through two objectives arranged in mirror-symmetrical fashion. The two objectives are arranged on different sides of their common object plane. At the object point, this interferometric illumination causes an interference pattern that, in a context of constructive interference, exhibits a principal maximum and several secondary maxima. This is referred to as “type A” 4-pi microscopy when only the excitation light interferes, and “type C” in the case of simultaneous interference of the detected light. With this double confocal scanning microscope, greater axial resolution than with a conventional scanning microscope can be achieved as a result of the interferometric illumination.
A resolution enhancement can be achieved both laterally and axially with a combination of STED and a double confocal arrangement.
Coherent anti-Stokes Raman scattering (CARS) microscopy is a technique that is becoming increasingly important. One great advantage is that the samples do not need to be labeled with dyes. In addition, living cells can be investigated.
As compared with conventional Raman microscopy and known confocal Raman microscopy, with CARS microscopy a higher detected light yield can be achieved, disruptive side effects can be better suppressed, and detected light can be more easily separated from the illuminating light. A detection pinhole is needed for conventional confocal Raman spectroscopy in order to achieve good axial resolution, as well as a high-resolution spectrometer. CARS, on the other hand, is a nonlinear optical process (four-wave mixing process). As in the case of multi-photon microscopy, in which two or more photons are absorbed simultaneously, a detection pinhole is not required because the probability of simultaneous in-phase coincidence of multiple photons is greatest at the focus, as a consequence of the higher photon density. Without a detection pinhole, the same axial resolution as in multi-photon microscopy is achieved. Two lasers emitting light of different wavelengths (νP and νs, the pump and Stokes lasers) are usually used for CARS spectroscopy; νs should be tunable in order to generate a CARS spectrum νCARS (νCARS=2νP−νS, ICARS≈(IP)2·IS). If the frequency difference νP-νs matches the frequency difference between two molecular vibration states |1 and |0 in the sample, the CARS signal is then in fact amplified further. For microscopic applications, the pump light beam and Stokes light beam are coaxially combined and focused together onto the same sample volume. The direction in which the anti-Stokes radiation is emitted is determined by the phase adaptation condition for the four-wave mixing process.
U.S. Pat. No. 4,405,237, “Coherent anti-Stokes Raman device,” discloses an apparatus in which two pulsed laser beams, generated by two lasers and having different wavelengths in the visible or UV region of the spectrum, are used to illuminate a sample simultaneously. With appropriate wavelength selection, the sample can be excited so that it emits the characteristic coherent anti-Stokes Raman radiation.
Just as in STED microscopy, it is important in CARS microscopy to be able to adjust both the time offset of the light pulses of the pump light beam and Stokes light beam, and the respective pulse widths over time, in terms of the sample being investigated.